Diagnostic assays are assays to test for the presence of an analyte in a test sample. Typically, diagnostic assays are immunoassays, that is, they involve the detection, of or exploit the use of, antibody molecules. Immunoassays are analytical tests that rely on the specificity of the reaction between antibodies and antigens to measure the concentration of either an antigen or an antibody in a sample of interest. In its simplest form, an immunoassay is the reaction of soluble antibody with soluble antigen and the product of the immunoassay is a measurement of the amount of antibody or antigen reacting with a known or constant amount of antigen or antibody, respectively. A variety of immunoassays are known in the art and more sophisticated immunoassays with increased assay sensitivity use magnifiers, amplifiers, or indicators, so that smaller quantities of antigen or antibody can be measured. The most commonly used immunoassays take advantage of the properties of certain enzymes. When the antibodies are linked to an enzyme, enzymatic activity serves as a marker for antibody binding.
Immunoassays can be accomplished using a variety of formats to meet different requirements such as sensitivity, simplicity, and/or feasibility of automation, and the like. These formats can be categorized into two general groups: heterogeneous or homogenous immunoassays. Heterogeneous systems are generally more sensitive, but usually require a series of washing steps to separate free from bound analyte (i.e., the molecule to be detected, whether antibody, antigen, or the like). Most commercially available immunoassay systems are heterogeneous because assays that are of high sensitivity, low background and low cost are preferred. The typical steps for performing a common heterogeneous immunoassay includes a coating step; that is, the immobilization of antigen or capturing antibodies onto a solid carrier. Next there are washing steps, incubation of the coated surface with sample, more washing steps, incubation with one or more detecting antibodies, signal amplification and development. The whole procedure takes several hours to several days to complete, and efficient automated systems are not available for these assays. High sensitivity for analyte in the assays is often achieved using sensitive enzymatic labels such as luciferase, alkaline phosphatase, galactosidase, and the like. Sensitive labels can reach a level of sample detection of less than 10.sup.-21 mol, with a potential to reach the limit of the antibody's affinity under optimized conditions (usually around 10.sup.-15 to 10.sup.-16 mol of analytes).
Homogenous immunoassays, on the other hand, are immunoassays where both the binding reaction and the detection and/or quantitation of the reaction are performed in solution without separating the free from the bound components. The sensitivity of the homogenous immunoassay is reduced as compared with a heterogeneous format. Homogenous immunoassays can be classified into two major types: competitive and noncompetitive binding immunoassays. Competitive assays usually include antigen labeled with enzyme or enzyme modulators (e.g., an inhibitor, activator, allosteric effector, or the like). The assay is typically based on a competitive reaction of antibody with antigen labeled with enzyme (conjugate) and antigen in present in the sample. The enzyme activity linked to the antigen is either inhibited or activated when the conjugate forms an immunocomplex with antibody. While it is relatively simple, these assays have several disadvantages. In general, the assays are insensitive as compared to heterogenous assays. Homogenous assay sensitivity depends on the concentration of the conjugate. In addition, homogenous assays can be inefficient at detecting high molecular weight antigens. On the other hand, antibody labeled with enzyme is used in noncompetitive binding assays based on either substrate "channeling", caused by close contact of two enzymes, or the modulation of enzyme activity due to binding of antigen to the conjugate. This format is more sensitive than a competitive assay; however, the assay format is still less sensitive than heterogeneous systems since, in general, either the concentration of the conjugate cannot be lower than a certain limit or the final signal detection is based on loss of activity rather than gain of activity. In addition, homogenous assays tend to have a higher noise to signal ratio than noncompetitive immunoassays.
Recent immunoassay developments focus on assay sensitivity, simplicity and assay automation. Homogenous systems are useful since multiple handling steps are typically not required making them particularly adaptable to automation. As noted, homogenous assay sensitivity is somewhat reduced as compared with heterogenous assay formats. For example, in homogenous immunoassays, an increase in assay sensitivity can be achieved by decreasing the amount of antigens or antibodies in the assay. Both antigen and antibody are labeled with enzyme in the reaction system but there is a level to which the antibody or antigen can be reduced and still generate a finctional assay. Most commercially available homogenous assays use competitive formats with sensitivities ranging in the microgram to nanogram levels. The analytes detected in these assays are limited to haptens and low-molecular-weight substances. Homogenous systems are not commercially available for high-molecular-weight proteins.